1. Field of the Invention
THIS INVENTION relates to the production of proteins, plasmids coding therefor and organisms containing such plasmids.
2. Background Information
It is known to modify microbes to produce desired proteins, for example enzymes, by incorporating plasmids coding for a desired protein into a microbe which would not otherwise produce it or which would not produce it in sufficient quantity. There is however a tendency for the plasmids to be lost on prolonged cultivation of the organism. It is believed that this is at least partly due to the production, on each division of the microbes, of a small number of daughter cells which contain none of the said plasmids and which are in consequence at a selective advantage over those which contain the desired plasmids. In the course of time, cells which contain none of the desired plasmids increase as a proportion of the total cells present.
In order to overcome this effect it is known to incorporate in the plasmid one or more genes which give a selective advantage to the microbe, for example genes giving resistance to an antibiotic are suitable if the microbes are cultivated in the presence of the antibiotic, or genes making good a deficiency in the host organism may be incorporated.
There is also a tendency for mutated variants of the plasmid which do not produce the desired protein to be produced for example by a mutation leading to the introduction of a stop codon in the gene or partial or complete deletion of the protein coding sequence. Microbes containing such mutated plasmids will tend to have a selective advantage compared with those having the original plasmid and the desired protein producing capability of the microbe may be reduced or lost on prolonged cultivation.
In DDR patent 233,851 A1 there are disclosed vector plasmids in which a sequence is present twice in opposite senses (inverted repeat sequences) into each of which sequences duplicate genes are cloned or recloned. This is said to cause increased synthesis of the gene product in the microorganism due to the gene dosage effect. The inverted repeat sequences must each have at least one homologous cleavage site into which the duplicate genes may be inserted. Stable plasmids are disclosed as producible.